Colicin E1 is a bacteriocin which has multiple functional states, existing both as a soluble protein and as a protein which can form a voltage-gated channel in a membrane. In vitro this conversion is driven by a pH change. An 18-20 kD fragment cleaved from the C-terminal end of colicin E1 retains all the channel forming properties of the whole molecule. crystals of this fragment have been obtained at two pHs which represent the soluble and the membrane form. The three-dimensional crystallographic structure of the high pH (soluble form) crystal will be solved. Preliminary studies indicated that these crystals diffract strongly to 2.2 A and are resistant to decay in the X-ray beam. Full native data will be collected to 2.2 A (Aims 1,7). Mercurial derivatives will be prepared with the single cysteine of the protein and with site-directed mutants having additional cysteines (Aim 2). Crystallographic data will be collected from these and other derivatives (Aims 3,4). Phases will be determined by single wavelength resolved-anomalous phasing, multiple isomorphous replacement, single isomorphous replacement/density modification or molecular replacement (Aim 5), and the three-dimensional structure built and refined (Aims 6,7). Crystallization of the intact colicin E1 molecule and improvements in the low pH (channel form) crystals will be made (Aims 8,10). It will also be determined if the conversion from the soluble to the membrane form can be done in the crystalline state (Aim 9). This work will lead not only to an understanding of the structural basis for these transitions in colicin E1 but may also shed light on the general mechanism of voltage-gated channels, protein translocation, and toxin action.